Neuron Reconstruction and Analysis Workshop
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The slides from the 2013 Neuron Reconstruction and Analysis Workshop presented by Dr. Julie Korich and Dr. Susan Tappan.
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Neuron Reconstruction and Analysis Workshop
- 1. Neuron Reconstruction and Analysis Workshop Julie Korich, Ph.D. Susan Tappan, Ph.D.
- 2. Workshop Outline • Neurolucida manual neuronal reconstructions • Tools for automatic neuronal reconstructions • AutoNeuron, AutoSpine and AutoSynapse modules • Imaging considerations • Morphometric analysis in Neurolucida Explorer • 3D Visualization of neuron reconstructions • Preview of Neurolucida 360 mbfbioscience.com
- 3. Introduction to Neurolucida • Reconstruction of neuronal structures • Quantify neuronal outgrowth in response to growth factors, drugs, etc. • Calculate spine and synaptic densities • Quantification of anatomical regions and cells • Calculate volume of infarct or tumor • Map stem cell migration in the spinal cord • Identification of neuronal networks and connectivity within an anatomical region mbfbioscience.com
- 4. LABELING IMAGING TRACING & RECONSTRUCTING ANALYZING Spectrum brightfield confocal two-photon Golgi Transgenic Transfection Injection/Fill EM Images Image stacks Virtual slides 2D/3D Neurolucida AutoNeuron AutoSpine AutoSynapse Neurolucida Explorer Blue Brain NeuroMorpho Whole Brain Biolucida NEURON Cholera Toxin .asc .dat .xml .obj Specificity mbfbioscience.com
- 5. Manual Neuron Reconstruction: • Directly on the scope • From images and image stacks mbfbioscience.com
- 6. Reconstructing Neurons Directly from Slides High resolution digital camera Microscope with high quality optics Motorized stage focus encoder, and stage controller Computer with MicroBrightField software and video capture card mbfbioscience.com
- 7. Reconstruct Neurons Directly from Slides (cont.) • The full extent of the dendrites and axons usually extend across multiple fields-of-view 150 serial sections • A motorized stage moves the specimen when tracing outside the field-of-view • The x,y,z information is stored to create a 3D reconstruction Courtesy of Dr. Rosa Cossart mbfbioscience.com
- 8. Reconstructing from Images • Load 2D images, 3D image stacks or montages into NL for 2D or 3D reconstruction • Trace through the entire stack or montage while focusing through the stack • Stacks can be acquired on a MBF system, on a confocal, or a 2-photon scope Image courtesy of MBF Bioscience mbfbioscience.com
- 9. Image Montage Module • • A number of overlapping image stacks were acquired that need to be aligned Image Montage Module will automatically align confocal stacks in XYZ Image Stacks Courtesy of Dr. Rosa Cossart mbfbioscience.com
- 10. Image Montage Module Image Stacks Courtesy of Dr. Rosa Cossart mbfbioscience.com
- 11. Adding Spines and Varicosities • Marked while tracing or once the dendrite is reconstructed • Use the spine toolbar to add spines • Use the marker tool bar to add varicosities or other features mbfbioscience.com
- 12. Reconstructing Anatomical Regions and Neurons • • • Trace contours across serial sections to reconstruct an anatomical region of interest, lesions, etc. Map neuronal projections and cells From live video or images collected throughout the ROI http://www.mbfbioscience.com/brain-mapping/cytoarchitectonics mbfbioscience.com
- 13. Changing Tracing Colors • Change the display of neurons, marker, and contours • Prior to Tracing: • Options>Display Preferences> Neuron, Marker, or Contour tab mbfbioscience.com
- 14. Editing • • While tracing, hit CTRL Z to delete the last point placed After tracing, use the editing tool to: • Modify fibers: • • • • • • Delete trees (fibers) Modify thickness along the tree Add branch points Modify colors Correct z errors Modify contours and markers • • • • Delete Modify thickness Resize Modify colors mbfbioscience.com
- 16. AutoNeuron module • • • Automatic reconstruction of neuronal processes and cell somas in 2D and 3D Uses fully automatic or interactive modes Recommend high magnification images with a small Z step (around 0.5µm) 20mm http://www.mbfbioscience.com/image-gallery mbfbioscience.com
- 17. AutoNeuron Advanced Options • Step 5 of the AutoNeuron workflow • Seed detection: • Adjust sampling density to ensure uniform sampling and seed coverage • Tracing: • AN sets the most optimal tracing settings based on the type of image: low magnification confocal, high magnification confocal and brightfield • Branch connections: • • Ignore traces shorter than user defined amount Adjust tolerance to gaps in staining mbfbioscience.com
- 18. DWORK Images courtesy of Dr. Andrew Dwork Images courtesy of Andrew Dwork mbfbioscience.com
- 19. AutoSpine Module: Spine Detection • Automated reconstruction of dendritic spines • • • Dendritic branch can be traced manually or automatically Dendritic spines modeled as a 3D mesh using defined parameters Recommend high magnification image stack with small Z step (under 0.5µm) Image courtesy of Dr. Jacob Jedynak mbfbioscience.com
- 20. AutoSynapse Module: Putative Synapse Detection • • • • Putative synapes automatically detected along a traced branch & modeled as a 3D mesh using defined parameters User determines detection distance from dendrite Recommend high magnification image stack with small Z step (under 0.5µm) Future versions will support colocalization Images courtesy of Dr. Francisco Alvarez & Travis Rotterman mbfbioscience.com
- 21. The Spine/Synapse Detector is a Toroid Outside radius Inside radius Image courtesy of Dr. Jacob Jedynak mbfbioscience.com
- 22. Editing • After tracing, use the editing tool as you would for manual traces • AutoNeuron: • The splice tool is most often used • AutoSpine: • Delete and classify spines • AutoSynapse • Delete synapses mbfbioscience.com
- 23. Orthogonal View for Editing • Displays portion of the image and tracing in Z • Make editing complex neurons easier Image courtesy of Dr. Jacob Jedynak mbfbioscience.com
- 24. Imaging for Reconstruction • Reconstruction goals • What to choose: • • • At the scope or from images? Time vs Effort Imaging modality • • Brightfield Fluorescence – CFM or MPFM Improve image analysis with correct capture and post-processing techniques. Lateral resolution Axial resolution Objective choice Depth of field Step size mbfbioscience.com
- 25. Axial Resolution Matters Image captured by MBF mbfbioscience.com
- 26. Axial resolution impacts reconstruction granularity Reconstruction courtesy of Bob Jacobs mbfbioscience.com
- 27. Tips for better reconstructions Brightfield: • Select: • • • • • • Coverglass (#1.5) Mounting medium Objective Immersion medium Koehler Illumination Fully open condenser If mapping live: • Place points often as you focus Image courtesy of Dan Peruzzi If imaging: • Use small step sizes (0.5 µm or less) • Create a virtual tissue mbfbioscience.com
- 28. Tips for better reconstructions Fluorescent: • Select: • • • • • • • • Coverglass (#1.5) mounting medium Objective Immersion medium Small step sizes (0.5 µm or less) Create a virtual tissue for seamless fields of view Maximize Dynamic Range After acquisition, deconvolve if necessary If using single or multiphoton microscope: • Match the Pinhole Size for each fluorophore! Image from Randy Bruno. Figure from Dumitru, Rodriguez and Morrison Nat Protoc. 2011 August 25; 6(9): 1391–1411. mbfbioscience.com
- 29. Adjust the dynamic range. Overexposure exaggerates axial blur. Image courtesy of Rosa Cosart
- 30. Note circular profile. Improve image analysis with correct capture and post-processing techniques. Image courtesy of Ryan Ash
- 31. Morphometric Analysis in Neurolucida Explorer MORPHOM3D VISUALIZATION AND mbfbioscience.com
- 32. Data analysis • Neuronal Analyses • Spine Data • Synapse Data http://vadlo.com/cartoons.php?id=71 mbfbioscience.com
- 33. Neuronal Analysis Branching analysis • Length per tree (dendrite/axon), per neuron, and per branch order Sholl Analysis • Calculated per tree and branch order Layer Analysis • Calculate length within cortical layers Branch Analysis • Calculate branch angles and numbers of branch points mbfbioscience.com
- 37. 3D Visualization Module • • Integrated within MBF software Display 3D rendering of objects built from reconstructions • • • • Rotate and zoom Place a “skin” around wireframe and adjust opacity Display the tracing and image data simultaneously Save solids view as a TIFF or JPEG2000 or create an animated movie for display (.avi) mbfbioscience.com
- 39. Future Directions – Neurolucida 360 & SpineStudio • Partnership with Dr. Patrick Hof and original developers of Neuron Studio • Full 3D interactive tracing and editing • Open API for 3rd party algorithm plug-ins mbfbioscience.com
- 40. Thanks! National Institutes of Health MBF Programmers, Staff, and Staff Scientists All of you for attending our workshop Current MBF Customers who provided the image data NIMH grants MH076188, MH085337, MH93011 mbfbioscience.com